ABSTRACT
Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25-36 of PrP corresponds to one antibody, and epitopes located in region 222-229, to the other two. The antibodies to fragment 222-229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.
Subject(s)
Antibodies, Monoclonal/immunology , Epitopes/immunology , Immunoglobulin M/immunology , Prions/immunology , Animals , Brain/immunology , Cattle , Encephalopathy, Bovine Spongiform/diagnosis , Encephalopathy, Bovine Spongiform/immunology , Enzyme-Linked Immunosorbent Assay , Epitope Mapping/methods , Immunization , Mice , Mice, Inbred BALB C , Prions/genetics , Recombinant ProteinsABSTRACT
The endogenous protein survivin is present in tumor cells and inhibits apoptosis. The influence of vaccination of mice by survivin fragments on growth of various types of tumors was studied to examine the possibility of creation of an antitumor vaccinating agent on its basis. Two peptides corresponding to the (118-144) and (80-88)-(153-165) sequences of survivin 2B were chosen and synthesized on the basis of literature data and theoretical calculations. Their ability to stimulate antibody production in mice of the C57BL/6J line (b haplotype) and in BDF1 hybrids (b x d haplotype) was investigated. Both peptides were shown to stimulate production of antibodies that bound the recombinant survivin in the BDF1 mice. Immunization of the BDF1 and C57BL/6J mice with the recombinant survivin resulted in the formation of antibodies that reacted with the (118-144) peptide. The effect of preventive vaccination with the peptides and the recombinant protein on the dynamics of growth of several species of tumors was studied. Vaccination with the (80-88)-(153-165) peptide was found to cause an antitumor effect in BDF1 mice suffering from sarcoma S-37. Thus, the creation of an antitumor agent on the basis of this peptide is a promising area of further studies.
Subject(s)
Microtubule-Associated Proteins/immunology , Neoplasm Proteins/immunology , Neoplasms, Experimental/prevention & control , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Antibody Formation , Epitopes, T-Lymphocyte , Humans , Immunotherapy , Inhibitor of Apoptosis Proteins , Mice , Mice, Inbred Strains , Microtubule-Associated Proteins/chemistry , Molecular Sequence Data , Neoplasm Proteins/chemistry , Neoplasms, Experimental/immunology , Peptide Fragments/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Species Specificity , Survivin , Xenograft Model Antitumor AssaysABSTRACT
The effect of immunization with the synthetic fragments of the alpha7 subunit of the acetylcholine nicotine receptor on the spatial memory of mice subjected to olfactory bulbectomy, which causes the development of the neuro-degenetrative disease of Alzheimer's type, was studied. Mice of the NMRI line were immunized with the KLH conjugates of two peptide fragments of the N-terminal fragment of the alpha7 subunit extraxcellular fragment, subjected to olfactory bulbectomy to cause the development of the neurodegenetrative disease of Alzheimer's type, and then the state of the spartial memory was evaluated. It was shown that 20% of bulbectomized mice immunized with the N-terminal 1-23 fragment exhibited good spatial memory after training. Immunization with the peptide construct (159-167)-(179-188) consisting of two hydrophilic exposed regions of alpha7-subunit induced good spatial memory in 50% of bulbectomized mice, while in the control group, which received only KLH, none of the animals were educated. Thus, the development of immunotherapy with peptide (159-167)-(179-188) seems to be a promising approach to prophylaxis and treatment of Alzheimer's disease.
Subject(s)
Alzheimer Disease/therapy , Immunization , Memory/drug effects , Protein Subunits/immunology , Receptors, Nicotinic/immunology , Alzheimer Disease/epidemiology , Alzheimer Disease/immunology , Animals , Hemocyanins/immunology , Hemocyanins/pharmacology , Humans , Mice , Protein Subunits/pharmacologyABSTRACT
Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).
Subject(s)
Antibodies, Monoclonal/biosynthesis , Peptide Fragments/immunology , Receptors, Nicotinic/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/blood , Antibody Affinity , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Molecular Sequence Data , Rats , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16- to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.
Subject(s)
Encephalitis, Tick-Borne/prevention & control , Peptides/immunology , Viral Nonstructural Proteins/immunology , Amino Acid Sequence , Animals , Antibodies, Viral/blood , Immunization , Mice , Mice, Inbred Strains , Molecular Sequence Data , Peptides/chemistry , Peptides/therapeutic use , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/therapeutic useABSTRACT
Potential B epitopes and T-helper epitopes in the N-terminal extracellular domain of the alpha7-subunit of human acetylcholine receptor (AChR) were theoretically calculated in order to reveal peptides that can induce the formation of specific antibodies to this domain. Four peptides structurally corresponding to four alpha7-subunit regions containing 16-23 aa and three of their truncated analogues were synthesized. Rabbits were immunized with both free peptides and protein conjugates of their truncated analogues, and a panel of antibodies to various exposed regions of the N-terminal extracellular domain of the AChR alpha7-subunit was obtained. All of the four predicted peptides were shown to induce the production of antipeptide antibodies in free form, without conjugation with any protein carrier. The free peptides and the protein conjugates of truncated analogues induced the formation of almost equal levels of antibodies. Most of the obtained antisera contained antibodies that bind to the recombinant extracellular N-terminal domain of the rat AChR alpha7-subunit and do not react with the analogous domain of the alpha1-subunit of the ray Torpedo californica AChR.
Subject(s)
Antibodies/immunology , Peptides/immunology , Receptors, Nicotinic/immunology , Amino Acid Sequence , Animals , Epitopes , Humans , Immunization , Molecular Sequence Data , Peptides/chemical synthesis , Protein Subunits/immunology , Rabbits , Rats , Torpedo , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
The antibodies to the bovine prion protein were produced by immunizing mice of three lines with five synthetic fragments of the protein and their six analogues. The analogues contained the amino acid substitutions that, according to theoretical calculation, should lead to an increase in the immunogenic activity of peptides. All the peptides, except for one, induced the formation of antibodies. All the sera containing the antipeptide antibodies were tested by an immunohistochemical method. The sera that were effectively bound to the brain preparations from the bovine with spongiform encephalopathy were identified; it was shown that they do not interact with the preparations of normal brain. Therefore, it was shown that the immunization of mice with the synthetic fragments of a prion protein helps obtain specific antibodies suitable for the study and diagnostics of prion diseases.
Subject(s)
Antibodies, Monoclonal/immunology , Brain/immunology , Encephalopathy, Bovine Spongiform/immunology , Peptide Fragments/immunology , Prions/immunology , Amino Acid Sequence , Amino Acid Substitution , Animals , Cattle , Immune Sera/immunology , Immunization , Immunoenzyme Techniques , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Species SpecificityABSTRACT
A simple method for the sequence prediction of peptides capable of the in vivo stimulation of antibody production in mice without conjugation with protein carriers was proposed on the basis of literature data on the structure of T-helper epitopes active in vivo. According to this approach, a potentially active peptide should contain a nine-membered sequence with a hydrophobic amino acid residue in the first position and a positively charged residue in the ninth position. The efficiency of this approach was confirmed by the presence of such sequences in the previously described synthetic peptides with immune activities, by the application of this approach to the choice of immunogenic fragments within the sequences of various proteins that exhibited further the specific activity, and by the construction of immunogenic peptides on the basis of inactive natural sequences.
Subject(s)
Antibody Formation/drug effects , Epitopes, T-Lymphocyte/immunology , Peptide Fragments/chemistry , Peptide Fragments/immunology , T-Lymphocytes, Helper-Inducer/immunology , Amino Acid Sequence , Animals , Immunization , Mice , Molecular Sequence Data , Peptide Fragments/pharmacology , Protein Conformation , Structure-Activity RelationshipABSTRACT
Four potentially immunoactive peptide fragments of the NspA protein from the outer membrane of the bacterium Neisseria meningitidis were synthesized in order to create a synthetic vaccine against the meningococcal infection by the serogroup B bacterium. Mice of various lines were immunized with the free peptides nonconjugated with a protein carrier. All the synthetic peptides were shown to induce the production of the antipeptide antibodies in mice. A peptide capable of inducing a decrease in the number of bacteria in blood and the protection of infected animals from death was found in the experiments on the protection of the animals infected with two strains of the Neisseria meningitidis serogroup B. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.